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I-crei meganuclease variants with modified specificity, method of preparation and uses thereof

a meganuclease and specificity technology, applied in the field of i-crei meganuclease variants with modified specificity, method of preparation and use thereof, can solve the problems of low efficiency, limited technology, and inability to have a natural meganuclease cleaving a gene of interes

Active Publication Date: 2012-10-11
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for preparing a mutated I-CreI meganuclease variant with a modified cleavage specificity. The method involves replacing certain amino acids in the I-CreI pdb accession code with different amino acids, and selecting I-CreI meganuclease variants that have a specific cleavage profile at certain positions in a double-strand DNA target. The mutated I-CreI meganuclease variant can be used for genetic engineering and molecular biology applications.

Problems solved by technology

Homologous gene targeting is the most precise way to stably modify a chromosomal locus in living cells, but its low efficiency remains a major drawback.
However, this technology is still limited by the low number of potential natural target sites for meganucleases: although several hundreds of natural homing endonucleases have been identified (Belfort and Roberts, 1997; Chevalier and Stoddard, 2001), the probability to have a natural meganuclease cleaving a gene of interest is extremely low.

Method used

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  • I-crei meganuclease variants with modified specificity, method of preparation and uses thereof
  • I-crei meganuclease variants with modified specificity, method of preparation and uses thereof
  • I-crei meganuclease variants with modified specificity, method of preparation and uses thereof

Examples

Experimental program
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Effect test

example 1

Screening for New Functional Endonucleases

[0141]The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity, are described in the International PCT Application WO 2004 / 067736. These assays result in a functional LacZ reporter gene which can be monitored by standard methods (FIG. 3a).

A) Material and Methods

a) Construction of Mutant Libraries

[0142]I-CreI wt and I-CreI D75N (or I-CreI N75) open reading frames (SEQ ID NO:69, FIG. 4A) were synthesized, as described previously (Epinat et al., N.A.R., 2003, 31, 2952-2962). Mutation D75N was introduced by replacing codon 75 with aac. The diversity of the meganuclease library was generated by PCR using degenerate primers from Sigma harboring codon VVK (18 codons, amino acids ADEGHKNPQRST) at position 44, 68 and 70 which interact directly with the bases at positions 3 to 5, and as DNA template, the I-CreI D75N gen...

example 2

I-CreI Meganuclease Variants with Different Cleavage Profiles

[0153]The validated clones from example 1 showed very diverse patterns. Some of these new profiles shared some similarity with the initial scaffold whereas many others were totally different. Various examples of profiles, including wild-type I-CreI and I-CreI N75, are shown in FIGS. 8 and 9a. The overall results (only for the 292 variants with modified specificity) are summarized in FIG. 7.

[0154]Homing endonucleases can usually accommodate some degeneracy in their target sequences, and one of our first findings was that the original I-CreI protein itself cleaves seven different targets in yeast. Many of our mutants followed this rule as well, with the number of cleaved sequences ranging from 1 to 21 with an average of 5.0 sequences cleaved (standard deviation=3.6). Interestingly, in 50 mutants (14%), specificity was altered so that they cleaved exactly one target. 37 (11%) cleaved 2 targets, 61 (17%) cleaved 3 targets and ...

example 3

Novel Meganucleases can Cleave Novel Targets While Keeping High Activity and Narrow Specificity

A) Material and Methods

a) Construction of Target Clones

[0156]The 64 palindromic targets were cloned into pGEM-T Easy (PROMEGA), as described in example 1. Next, a 400 bp PvuII fragment was excised and cloned into the mammalian vector pcDNA3.1-LACURAZ-ΔURA, described previously (Epinat et al., precited). The 75 hybrid targets sequences were cloned as follows: oligonucleotides were designed that contained two different half sites of each mutant palindrome (PROLIGO).

b) Re-Cloning of Primary Hits

[0157]The open reading frame (ORF) of positive clones identified during the primary screening in yeast was recloned in: (i) a CHO gateway expression vector pCDNA6.2, following the instructions of the supplier (INVITROGEN), and ii) a pET 24d(+) vector from NOVAGEN Resulting clones were verified by sequencing (MILLEGEN).

c) Mammalian Cells Assay

[0158]CHO-K1 cell line from the American Type Culture Collect...

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Abstract

Method of preparing I-CreI meganuclease variants having a modified cleavage specificity, variants obtainable by said method and their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes. Nucleic acids encoding said variants, expression cassettes comprising said nucleic acids, vectors comprising said expression cassettes, cells or organisms, plants or animals except humans, transformed by said vectors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. Ser. No. 12 / 859,905, filed Aug. 20, 2010, which is a continuation of U.S. Ser. No. 11 / 908,798, filed on Sep. 17, 2007, which is a 35 U.S.C. §371 National Stage patent application of PCT / IB2006 / 001203, filed on Mar. 15, 2006, which claims priority under 35 U.S.C. §120 and 35 U.S.C. §365(c) to International patent application PCT / IB2005 / 003083, filed on Sep. 19, 2005, and also claims priority under 35 U.S.C. §120 and 35 U.S.C. §365(c) to International patent application PCT / IB2005 / 000981, filed Mar. 15, 2005.BACKGROUND OF THE INVENTION[0002]The present invention relates to a method of preparing I-CreI meganuclease variants having a modified cleavage specificity. The invention relates also to the I-CreI meganuclease variants obtainable by said method and to their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes.[000...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/16C12N15/63C12N1/21A01K67/027C12N1/19C12N15/55A01H5/00
CPCA61K38/00C12N9/22A61K48/00A61P31/00A61P31/12A61P43/00
Inventor DUCHATEAU, PHILIPPEPAQUES, FREDERIC
Owner CELLECTIS SA
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